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Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases Cinya Chung, and Hung-Wen Li J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja401626t • Publication Date (Web): 29 May 2013 Downloaded from http://pubs.acs.org on June 2, 2013
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Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases Cinya Chung, and Hung-Wen Li* Department of Chemistry, National Taiwan University, Taiwan
KEYWORDS: DNA helicase, single-molecule observation. ABSTRACT: The heterotrimeric E. coli RecBCD enzyme composes of two helicase motors with different polarities: RecB (3’-to5’) and RecD (5’-to-3’). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (> 200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3’-to-5’ or 5’-to-3’ ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5’-to-3’ and 3’-to-5’) of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entered into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.
1. Introduction DNA helicases are a group of enzymes that unwinds duplex DNA by coupling with the nucleoside triphosphate hydrolysis. By generating ssDNA strands, helicases initiates essential DNA metabolic processes of replication, recombination and repair. In the process of generating ssDNA, it requires the enzyme to not only unwind duplex DNA but also translocate along the DNA substrate to achieve processive catalysis. In addition to duplex DNA unwinding, helicases have been implicated in displacing proteins from DNA substrates1-3. One of the recent proposed models for helicase motion is that helicases function as ssDNA translocases4,5. In this ssDNA translocase model, helicases moves along ssDNA to separate two strands of DNA to achieve unwinding, and to displace proteins bound to the DNA, and to generate ssDNA available for downstream biochemical reactions. The Escherichia coli RecBCD is a processive helicase which initiates homologous recombinational repair. This superfamily 1 (SF1) heterotrimeric enzyme is composed of three subunits, RecB (3’-to-5’ helicase), RecC, and RecD (5’-to-3’ helicase), which enables the enzyme to move along DNA unidirectionally. In double-strand-break (DSB) repair, RecBCD initiates the process by recognizing the nearly blunt-end damaged DNA, and processes this DNA by unwinding DNA strands using its motors6,7. Here we used RecBCD helicase as a model system to investigate the ssDNA translocation mechanism of a helicase. We used single-molecule tethered particle
motion (TPM) experiments8 to directly observe the ssDNA translocation activities of RecBCD in real-time. We have constructed DNA substrates which contains a long ssDNA (~200 nt) with no obvious secondary structure between duplex regions. As the bead-labeled enzyme translocates along the ssDNA substrate, the Brownian motion amplitude of the bead decreases as the DNA tether length reduces. We demonstrated that RecBCD helicase contains both 3’-to-5’ and 5’-to-3’ ssDNA translocase activities using single-molecule TPM methods. 2. Results 2.1 RecBCD translocates along long ssDNA gap in both polarities Purified biotinylated RecBCD enzymes attached with 200 nm streptavidin beads were found to successfully translocate along 907 bp fully dsDNA. At 30 µM ATP, the averaged translocation rate is 60 ± 15 bp/s (Brownian motion amplitude change: 5.6 ± 1.5 nm/s, N=27, Fig. S1), consistent with the previous report9. To directly test the ssDNA translocase activity of RecBCD helicases, we constructed the DNA substrates containing a long extended, secondary structure free ssDNA gap (details in Materials and Methods and Supporting Information). Control experiments using restriction enzyme digestion confirmed the existence of ssDNA gaps using the TPM setup (Fig. S2).
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A
B
C
Figure 1. Observation of individual RecBCD helicases translocating along ssDNA using a tethered particle motion experiment. (A) The DNA substrates contain a ~200 nt long, unstructured ssDNA gap flanked by a double-stranded DNA end for RecBCD loading and was linked to the surface through a ~500 bp dsDNA. Biotinylated RecBCD enzymes were attached to streptavidin beads visible under the optical microscope. Translocation occurred as the enzyme recognized the blunt-end DNA, and started unwinding. Brownian motion (BM) amplitude of the bead decreased as the enzyme translocated along DNA towards the surface. (B)-(C) Successful exemplary traces were observed as the enzymes translocated along the ssDNA region, across the ss/dsDNA junction, and to the surface end of the substrate (representative traces). Dashed lines referred to the BM amplitude at ss/dsDNA junction following the ssDNA gap. Both DNA substrates containing either 3’-to-5’ ssDNA (substrate 789, B) or 5’-to-3’ ssDNA (substrate 860, C) showed successful enzyme translocation along an ssDNA region. Experiments were performed under the ATP concentration of 30 µM in the presence of ATP regeneration system.
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The substrate 789 contains a 195 nt long ssDNA gap, and is annealed from three ssDNA segments. This substrate is immobilized on the anti-digoxigenin decorated coverglass through the 5’-labeled digoxigenin end (Fig. 1B). Since RecBCD recognizes the blunt-end DNA substrates, the helicase enters through the 66 bp fully dsDNA end and will translocate along the ssDNA in the 3’-to-5’ polarity towards the surface. When bead-coated RecBCD molecules were flown into the reaction chamber in the presence of ATP, the enzyme started translocating as it bound to the end of DNA substrate. Bead BM time trace showed smooth movement along the DNA substrate from the blunt entry end, translocating through the long ssDNA gap, to the ss/dsDNA junction, continuing onto fully dsDNA section until finishing unwinding/translocating all DNA substrate, and eventually dissociating from the surface (Fig. 1B). No pauses were observed among all full translocation traces (N=22), giving a similar pattern as in the case of translocating along fully duplex DNA. The successful translocation across long 3’-to-5’ ssDNA is a direct evidence that RecBCD helicase posseses a 3’-to-5’ ssDNA translocase activity10. Considering that RecB is the 3’-to-5’ helicase10, it is reasonable to suggest that the 3’-to-5’ translocation is attributed to the ssDNA translocase activity of the RecB motor. Within the experimental resolution, no apparent change in translocation speed was observed. Our proposal of RecB as a 3’-to-5’ ssDNA translocase is consistent with previous biochemical studies11,12 that RecBC with only one ATPase motor can cross a 3’-to-5’ gap. We also prepared the DNA substrate containing an ssDNA gap in the opposite direction that RecBCD will translocate in the 5’-to-3’ direction (substrate 860, Fig.1C). As shown in the exemplary BM time traces, RecBCD successfully translocated along this substrate, and were capable of moving along the ~500 bp dsDNA after the ss/dsDNA junction to the end of the surface-immobilized DNA (N = 33). Therefore, we concluded that RecBCD also possesses a 5’-to-3’ ssDNA translocase activity. Experiments including single-stranded DNA binding (SSB) proteins return with similar observation (Fig. S10). To verify the position of ss/dsDNA junction at these substrates containing the ssDNA gap, we carried out series of control experiments by putting biotin label at the junction location (Fig. S3A). Control experiments showed the Brownian motion amplitude at the ss/dsDNA junction with 50.4 ± 3.6 nm for substrate 789 and 54.7 ± 5.3 nm for substrate 860 (dashed lines, Fig. 1B and C). The Brownian motion for full length substrate 789 and 860 are 55.2 ± 5.0 nm and 65.0 ± 8.0 nm respectively. Brownian motion for the gapped substrates were found to be smaller than duplex DNA with the same size, likely due to the fact that ssDNA is much more flexible (with a persistence length < 2 nm13-15) than that of dsDNA (persistence length ~45-50 nm16,17). From the time traces, we could tell that the tether lengths had reduced as RecBCD translocated along the ssDNA region and across the junction. Translocation rates were determined by linear fitting of the Brownian time traces: Substrate 789 (N = 22), 3’-to-5’ translocation, average rate = 5.0 ± 2.0 nm/s; substrate 860 (N = 33), 5’-to-3’ translocation, average rate = 4.7 ± 1.7 nm/s (Fig. S4, at 30 µM ATP). The wide distribution of the histogram is consistent with previous studies, and is likely due to the enzyme’s heterogeneity18,19. The rates in either ssDNA polarilities are indistinguishable, and are similar to the duplex DNA unwinding rate, 5.6 ± 1.5 nm/s. This similarity in rates could be attributed to the limiting
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ATP concentration used here (
